Superscript 2 reverse transcriptase kit

Approximately 2 μg of total RNA from non-transgenic and transgenic jute seedlings was used for first-strand cDNA synthesis, followed by gene specific PCR. cDNA was prepared in a 20 μL reaction volume, which contained 2 μL of reverse transcriptase (200 U/μL), using SuperScript^TM II reverse transcriptase kit (Invitrogen, USA) according to the manufacturer’s manual. The reaction was carried out with an initial incubation at 50 °C for 50 min, then 5 min at 85 °C to inactivate the enzyme. Next RNAse H was added followed by an incubation at 37 °C for 20 min to remove the cDNA-RNA hybrid. 1 μL of the cDNA sample was used as a template for PCR amplification with COMT GSP forward/COMT GSP reverse and C4H GSP forward /C4H GSP reverse primers (Supplementary Table 1) to amplify the respective samples. PCR of the same cDNA samples was carried out using gene-specific primers for actin to be used as a loading control in gel electrophoresis. All PCRs included 30 cycles of denaturation at 95 °C for 30 s, 40 s at the optimal temperature for each gene specific primer set (Supplementary Table 1) for annealing and 40 s at 72 °C for extension. RT-PCR band intensities were quantified using the ImageJ software (http://rsbweb.nih.gov/ij/index.html).

Total mRNA was purified using the QuickPrep Micro mRNA purification kit from Pharmacia. cDNAs were synthesized using the SuperScriptTMII Reverse Transcriptase kit (Invitrogen) with 100 ng of mRNA and 2 pmol of Oligo(dT)₁₈ primer.

We used TRIzol reagent (Invitrogen, Carlsbad, USA) to extract the total RNA endometrial carcinoma tissues and corresponding healthy tissues. The SuperScriptTM II Reverse Transcriptase Kit (Invitrogen) was used to reversely transcript of 5 μg of total RNA. SYBRGreen reagent (Thermo Fisher Scientific) was used for quantitative real time polymerase chain reaction (qRT-PCR). For qRT-PCR, primers that were used to estimate the expression of total LOC134466 and TAC1 were listed in Table 2. The expression of these genes was normalized to that of GADPH. Besides, subsequent to the real-time RT-PCR, we examined the dissociation reaction diagram and then verified the specificity of the PCR. It is worth conducting all the experiments in triplicate.

Total RNA was extracted from DRGs using peqGOLD TriFast (peqlab) following the manufacturer’s protocol. RNA was determined with a peqlab NanoDrop ND-1000. Complementary DNA was synthesized from 5 μg of total RNA using the SuperScript TM II Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA) with oligo(dT) primers, according to the manufacturer’s protocol. One microlitre of 1:5 dilution cDNA was used in quantitative reverse transcriptase–PCR experiments using ABsolute QPCR SYBR Green ROX Mix (Thermo Scientific) on an ABI 7500 Real Time PCR System (ABS/Life Technologies). Primers used were as follows: Gabrb3 Forward: 5′-GCCAGCATCGACATGGTTTC-3′; Reverse: 5′-GCGGATCATGCGGTTTTTCA-3′. GAPDH Forward: 5′-ACCCTGTTGCTGTAGCCGTATCA-3; Reverse: 5′-TCAACAGCAACTCCCACTCTCCA-3.

The immature GV oocytes from donkey ovaries and in vitro matured MII oocytes were collected with 8 µL cell lysis buffer (cells-to-cDNA^TM II kit, Ambion, Shanghai, China). The total RNA was extracted and reverse transcribed to cDNA using the SuperScript^TM II Reverse Transcriptase Kit (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. In brief, the 20 µL mixture of reverse transcription system contained 8 µL of cell lysis buffer with six oocytes, 1 µL of DNase, 1.3 µL of DNase I buffer, 1 µL of EDTA, 1 µL of dNTP, 2 µL of random primer, 4 µL of 5 × first-stand buffer, 0.5 µL of RNase inhibitor, 2 µL of DTT, and 0.25 µL of FS RT (reverse transcriptase) enzyme.
Before quantitative real-time polymerase chain reaction (qRT-PCR) amplification, the primers of reference gene ACTB and target genes (GDF9, BMP15, LGALS3, and ALG5) were designed using Oligo 7.0 software (Table S1), and all primer synthesis work was done by GenSys Biotech (Nanning, China). The relative expression of all genes was quantified using the SYBR qPCR Master Mix (Vazyme, Shanghai, China) on the basis of the manufacturer’s instructions. Fluorescence data were acquired using a fluorescence ratio PCR instrument (Roche, Shanghai, China). Finally, the relative gene expression was calculated using the 2^−△△Ct method.

Fresh leaves of European mistletoe plant were ground to a fine powder in liquid nitrogen by using mortar and pestle. The RNA extraction was performed by using ISOLATE II RNA plant kit (Meridian Bioscience, London, UK) following the manufacturer’s instructions. The quality of the extracted RNA was later assessed on agarose gel electrophoresis containing formaldehyde and NanoDrop2000 (Thermo Fisher scientific, Waltham, MA, USA). The cDNA was reverse transcribed using SuperScriptTM-II reverse transcriptase kit (Invitrogen, Waltham, MA, USA) following the manufacturer’s instructions.

Total RNA from the four GC cell lines was isolated using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA), according to manufacturer’s instructions. Following spectrophotometric assessment of RNA yield and quality, 5 µg of each cell line’s total RNA were reverse-transcribed into single-stranded cDNA using Superscript^TM II Reverse Transcriptase kit (Invitrogen, Thermo Fisher Scientific), following manufacturer’s recommendations. Glycosyltransferase mRNA expression levels were quantified by RT-qPCR using TaqMan^TM Universal PCR Master Mix II, no UNG, and the specific TaqMan^TM gene expression assays, listed below, in a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA; Thermo Fisher Scientific). The mRNA expression levels of 18S Ribosomal 5 (RNA18S5) endogenous control were also measured for normalization of target gene abundance. RT-qPCR was carried out in triplicate for each biological sample and in duplicate for the negative controls. The following TaqMan^TM gene expression assays were used: FUT3 (Hs01868572_s1), FUT8 (Hs00189535_m1), MGAT3 (Hs02379589_s1), MGAT5 (Hs01073268_m1), ST3GAL3 (Hs00544035_m1), ST3GAL4 (Hs00920871_m1), ST6GAL1 (Hs00949382_m1), B3GALT5 (Hs00707757_s1), and RNA18S5 (Hs99999901_s1). Data were analyzed by ΔΔC_t method [60 (link)].